The present invention relates to a process for preparing an environmentally compatible porous material comprising beneficial nematodes with insecticidal activity, and to the biotic preparations produced therefrom. The environmentally compatible porous material may be directly applied to the desired target to eliminate the recovery procedures and formulation steps involved in currently used methods.
Nematode worms (Class Nematoda) have round elongated bodies that usually taper at the ends to almost a point. Unlike flat worms (Playtyhelminthes), nematodes have no cillia, the body being enclosed in a cuticle coat which, combined with the absence of circular muscles, severely limits the types of movements the nematodes can make, thus they appear to thrash about in an almost random fashion. Nematodes are extremely abundant, and occur in almost every type of habitat, with many free-living in soil or water, and are generally microscopic in size. Certain parasitic nematodes, of both plants and animals, can attain a length of up to one meter. Nematodes are continuous feeders, requiring an essentially unlimited source of food as they lack the ability to store food. (see generally Keeton (1980) Biological Science 3.sup.rd Edition, WW Norton & Co., New York).
The nematode-based pesticide of the present invention is not a nematicide (i.e. toxic to nematodes) but a kind of novel pesticide/biotic preparation which comprises beneficial nematodes associated with symbiotic bacteria in a biodegradable matrix which is useful for controlling pests that would harm cultivated plants. A nematode-based pesticide of the present invention has several advantages over typical chemical pesticides. For instance, general chemical pesticides are highly toxic, pesticide poisonous, and easy to result in environmental pollution; they affect natural predators, destroy ecological balance, and often cause the resistance against the targeted pests.
U.S Pat. No. 4,334,498 (this and all subsequent patent documents are hereby incorporated by reference in their entirety) disclosed a method for rearing nematodes using an assemblage for rearing nematodes comprises a growth medium suitable for the culture of the nematodes, the growth medium being dispersed to allow free movement of fluids over the surface of the medium and to maximize the ratio of the surface area of the medium to volume of the assemblage. The assemblage may comprise an animal tissue homogenate as growth medium dispersed over the surface of elements of inert material such as wood-wool or crumbed plastics foam.
U.S Pat. No. 4,334,498 describes that infective larvae of Neoaplactana carpocapsae have been observed to destroy a wide range of insect pests under laboratory conditions. This ability is accounted for by the nematode's own pathogenicity towards the insects, and by its association with the symbiotic bacterium, Achromobacter nematophilus, which is usually to be found in the intestinal lumen of infective species. Following ingestion by an insect, the nematode will usually penetrate the gut wall to enter the haemocoel and release A. nematophilus which mutliply and lead to the death of the host by septicaemia. Additionally, there will often be reproduction of the nematodes in the dead host and hence generation of further infective organisms. The patent discloses that infective nematodes will remain active for substantial periods of time in the absence of free water, provided their body moisture, oxygen supply and mobility can be maintained, and that this can be achieved by use of oil instead of water as a medium in which to store or disperse the nematodes. Thus the commonly practiced techniques for rearing nematodes are mostly based upon solid and liquid culture.
The compositions and method of the instant invention teaches a process for preparing nematode-based pesticide suitable for mass production and the formulation of preparations which will be suitable for long-term storage, for example for a time of more than six months. Because nematodes are multicellular animals, the preparations currently practiced are not suitable for prolonged storage, such as would be possible with other microbiotic preparations (such as storage for one year or more). It is recognized in the art that it would be most useful to have preparations of nematodes which could be stored for a period of time prior to use. For instance, WO 89/04602 and U.S. Pat. No. 4,615,883 describe a method and composition for an insecticide comprising a hydrated hydrogel capsule containing an insecticidally effective amount of at least one nematode capable of infecting an insect host, which capsule has sufficient hydration to maintain the viability and infectivity of said nematode. Also disclosed are methods for providing said insecticidal compositions. The method disclosed utilizes alginate to embed nematodes, and such preparations can be used for 3 to 6 months. However, one difficulty in using preparations such as this, is that it has to dissolved in sodium citrate (taking about 30 minutes), and then diluted with water before spraying onto crops.
The methods described in U.S. Pat. No. 4,334,498 and WO 88/08668 use the principle of nematode cryptobiosis, to carry out the dehydration of nematodes under conditions for decreasing relative humidity in a stepwise manner, to make the nematode preparations into essentially clays. The storage of the clay obtained may be suitable for 3 to 6 months under refrigerated conditions. U.S. Pat. No. 5,042,427 describes methods to transport entomopathogenic nematodes. It is described how it is necessary to store them in a manner such that a significant proportion of them survive after being stored and are reactivated when dispersed in water. The process provides storage by mixing an aqueous cream of clean third stage infective juveniles (J3) of nematodes with clay. The clay may be in chip form, or calcined, milled and sieved, but comporises from about 33 percent (by weight) to 67 percent (by weight) of the homogeneous mixture.
However, the clay process is commercially expensive to practice as it spends a great deal of time and energy in the dehydration procedure. Unfortunately, the clays must be extensively pretreated prior to use, as they have to be treated at high humidity (95% relative humidity) overnight to rehydrate the nematodes. In addition, the clays produced by this procedure, and the dust associated with these products are harmful to the lungs of humans.
U.S. Pat. No. 5,113,799 discloses an apparatus for the rearing of insects and the production of insect related products, primarily insect viruses and parasites commonly used for the control of insect pests. The apparatus comprises an environmental control system, a sealed enclosure consisting of a hood and a base, and a system of vertically supported food leaves.
U.S. Pat. No. 5,183,950 relates to methods to desiccate, package, store, and ship insect parasitic nematodes in both large and small quantities while maintaining their viability and pathogenicity to insects. In particular, to maintain the infective juveniles of the Steinernematid and Heterorhabditid nematodes in a state of dormancy so that their food reserves are not used up, and so that upon return to suitable conditions they revive and remain pathogenic to the insect host. In short, the methods and containers disclosed are designed to maintain the infective juveniles in a "cryptobiotic" state--a state of dormancy in which metabolism is suppressed. Several ways of doing this, with varying degrees of success, are +PG,6 known for organisms in general. The most generally suggested method and perhaps the most universally applicable is the induction of cryobiosis, i.e., reduced metabolism at low, usually freezing temperatures. In addition, and more difficult to achieve, are anhydrobiosis, which is induced by evaporative desiccation and the closely related osmobiosis, which is induced by osmotic desiccation.
PCT Application WO 85/03412 suggests methods of transport and storage which depend on maintaining putative anaerobic conditions and the presence of an antimicrobial agent. High osmotic strength solutions are also used to prevent bacterial growth. The proposed storage conditions also include an adsorbent such as charcoal or synthetic resins, although it is not clear what these agents are expected to adsorb. The disclosure exemplifies the use of formaldehyde as an antimicrobial, and proposes storage containers which contain both the nematodes and adsorbent charcoal.
T. N. Wang et al in Sugar Research Institute (1992) had reported that they utilize sugar dregs to absorb nematode infected liquid and apply them to the field. (The nematodes of this preparation may be stored at room temperature for about one month.) However, the use of bagasse as the matrix for culturing nematodes (adding culture medium) is not disclosed in the article. In addition, the bagasse will be easily attacked by fungi due to sugar residue.
Formulation and application technology in "Entomopathogenic Nematodes in Biological Control" Georgis. R. describes that vermiculite and peat can be used as moistened carriers for transporting and storing nematodes. However, the nematodes are taught to be cultured and recovered prior to applying vermiculite and peat. As described above, the recovery step is cost-consuming, and loss in nematode yield and the problem of waste water are great disadvantages to this method of transport. In addition, Georgis did not use vermiculite or peat as support medium for culturing nematodes.
Thus the present methods for preparation of nematode based insecticide preparations are cumbersome and labor-intensive, and do not afford effective long-term storage of viable materials. In contrast, the present invention teaches simplified and efficient methods for producing biodegradeable and bio-compatible biotic constructs useful for the long-term storage of nematode/bacterial preparations, and useful for the simplified application to subject plants.